Analysis of the phosphoproteome of the multicellular bacterium Streptomyces coelicolor A3(2) by protein/peptide fractionation,phosphopeptide enrichment and high‐accuracy mass spectrometry

The serine (Ser)/threonine (Thr)/tyrosine (Tyr) phosphoproteome of exponentially growing Streptomyces coelicolor A3(2) was analysed using the gel‐free approaches of preparative IEF for protein fractionation, followed by strong cation exchange peptide fractionation and phosphopeptide enrichment by TiO₂ metal oxide affinity chromatography. Phosphopeptides were identified using LC‐ESI‐LTQ‐Orbitrap^? MS. Forty‐six novel phosphorylation sites were identified on 40 proteins involved in gene regulation or signalling, central metabolism, protein biosynthesis, membrane transport and cell division, as well as several of unknown function. In contrast to other studies, Thr phosphorylation appeared to be preferred, with relative levels of Ser, Thr and Tyr phosphorylation of 34, 52 and 14%, respectively. Genes for most of the 40 phosphorylated proteins reside in the central “housekeeping” region of the linear S. coelicolor chromosome, suggesting that in general Ser, Thr and Tyr phosphorylation play a role in regulating essential aspects of metabolism in streptomycetes. A greater number of regulators and putative regulators were also identified compared with other bacterial phosphoproteome studies, potentially reflecting the complex heterotrophic and developmental life style of S. coelicolor. This study is the first analysis of the phosphoproteome of a member of this morphologically complex and industrially important group of microorganisms.     

Phosphorylation and activation of protein tyrosine phosphatase (PTP) 1B by insulin receptor

We have previously reported a direct in vivo interaction between the activated insulin receptor and protein-tyrosine phosphatase-1B (PTP1B), which leads to an increase in PTP1B tyrosine phosphorylation. In order to determine if PTP1B is a substrate for the insulin receptor tyrosine kinase, the phosphorylation of the Cys 215 Ser, catalytically inactive mutant PTP1B (CS-PTP1B) was measured in the presence of partially purified and activated insulin receptor. In vitro, the insulin receptor tyrosine kinase catalyzed the tyrosine phosphorylation of PTP1B. 53% of the total cellular PTP1B became tyrosine phosphorylated in response to insulin in vivo. Tyrosine phosphorylation of PTP1B by the insulin receptor was absolutely dependent upon insulin-stimulated receptor autophosphorylation and required an intact kinase domain, containing insulin receptor tyrosines 1146, 1150 and 1151. Tyrosine phosphorylation of wild type PTP1B by the insulin receptor kinase increased phosphatase activity of the protein. Intermolecular transdephosphorylation was demonstrated both in vitro and in vivo, by dephosphorylation of phosphorylated CS-PTP1B by the active wild type enzyme either in a cell-free system or via expression of the wild type PTP1B into Hirc-M cell line, which constitutively overexpress the human insulin receptor and CS-PTP1B. These results suggest that PTP1B is a target protein for the insulin receptor tyrosine kinase and PTP1B can regulate its own phosphatase activity by maintaining the balance between its phosphorylated (the active form) and dephosphorylated (the inactive form) state.     

Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes. We have recently reported the first in-depth phosphorylation site-resolved dataset from the model Gram-positive bacterium, Bacillus subtilis, showing that Ser/Thr/Tyr phosphorylation is also present on many essential bacterial proteins. To test whether this modification is common in Eubacteria, here we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site-resolved database of bacterial phosphoproteins to date (available at www.phosida.com) and used it to study evolutionary conservation of bacterial phosphoproteins and phosphorylation sites across the phylogenetic tree. We demonstrate that bacterial phosphoproteins and phosphorylated residues are significantly more conserved than their nonphosphorylated counterparts, with a number of potential phosphorylation sites conserved from Archaea to humans. Our results establish Ser/Thr/Tyr phosphorylation as a common posttranslational modification in Eubacteria, present since the onset of cellular life.     

Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor

PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B contains this motif (RYRDVS(50)), and wild-type PTP1B (but not mutants with substitutions for Ser(50)) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3(IR) cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with [(32)P]-orthophosphate. Insulin stimulation caused a significant increase in phosphorylation of wild-type PTP1B that could be blocked by pretreatment of cells with wortmannin or cotransfection of a dominant inhibitory Akt mutant. Similar results were observed with endogenous PTP1B in untransfected HepG2 cells. Cotransfection of constitutively active Akt caused robust phosphorylation of wild-type PTP1B both in the absence and presence of insulin. By contrast, PTP1B-S50A did not undergo phosphorylation in response to insulin. We tested the functional significance of phosphorylation at Ser(50) by evaluating insulin receptor autophosphorylation in transfected Cos-7 cells. Insulin treatment caused robust receptor autophosphorylation that could be substantially reduced by coexpression of wild-type PTP1B. Similar results were obtained with coexpression of PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an impaired ability to dephosphorylate the insulin receptor. Moreover, cotransfection of constitutively active Akt significantly inhibited the ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the insulin receptor. We conclude that PTP1B is a novel substrate for Akt and that phosphorylation of PTP1B by Akt at Ser(50) may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.     

The two faces of Janus: virulence gene regulation by CovR/S in group A streptococci

The group A streptococcus (GAS) causes a variety of human diseases, including toxic shock syndrome and necrotizing fasciitis, which are both associated with significant mortality. Even the superficial self-limiting diseases caused by GAS, such as pharyngitis, impose a significant economic burden on society. GAS can cause a wide spectrum of diseases because it elaborates virulence factors that enable it to spread and survive in different environmental niches within the human host. The production of many of these virulence factors is directly controlled by the activity of the CovR/S two-component regulatory system. CovS acts in one direction as a kinase primarily to activate the response regulator CovR and repress the expression of major virulence factors and in the other direction as a phosphatase to permit gene expression in response to environmental changes that mimic conditions found during human infection. This Janus-like behaviour of the CovR/S system is recapitulated in the binding of CovR to the promoters that it directly regulates. Interactions between different faces of the CovR DNA binding domain appear to depend upon DNA sequence, leading to the potential for differential regulation of virulence gene expression.     

The Eukaryotic-Like Ser/Thr Kinase PrkC Regulates the Essential WalRK Two-Component System in Bacillus subtilis

Most bacteria contain both eukaryotic-like Ser/Thr kinases (eSTKs) and eukaryotic-like Ser/Thr phosphatases (eSTPs). Their role in bacterial physiology is not currently well understood in large part because the conditions where the eSTKs are active are generally not known. However, all sequenced Gram-positive bacteria have a highly conserved eSTK with extracellular PASTA repeats that bind cell wall derived muropeptides. Here, we report that in the Gram-positive bacterium Bacillus subtilis, the PASTA-containing eSTK PrkC and its cognate eSTP PrpC converge with the essential WalRK two-component system to regulate WalR regulon genes involved in cell wall metabolism. By continuously monitoring gene expression throughout growth, we consistently find a large PrkC-dependent effect on expression of several different WalR regulon genes in early stationary phase, including both those that are activated by WalR (yocH) as well as those that are repressed (iseA, pdaC). We demonstrate that PrkC phosphorylates WalR in vitro and in vivo on a single Thr residue located in the receiver domain. Although the phosphorylated region of the receiver domain is highly conserved among several B. subtilis response regulators, PrkC displays specificity for WalR in vitro. Consistently, strains expressing a nonphosphorylatable WalR point mutant strongly reduce both PrkC dependent activation and repression of yocH, iseA, and pdaC. This suggests a model where the eSTK PrkC regulates the essential WalRK two-component signaling system by direct phosphorylation of WalR Thr101, resulting in the regulation of WalR regulon genes involved in cell wall metabolism in stationary phase. As both the eSTK PrkC and the essential WalRK two-component system are highly conserved in Gram-positive bacteria, these results may be applicable to further understanding the role of eSTKs in Gram-positive physiology and cell wall metabolism.     

JNK regulates cell migration through promotion of tyrosine phosphorylation of paxillin

The adaptor protein paxillin plays an important role in cell migration. Although the c-Jun amino-terminal kinase (JNK) phosphorylation of paxillin on Ser 178 has been found to be critical for cell migration, the precise mechanism by which JNK regulates cell migration is still not very clear. Here, the migration of human corneal epithelial (HCE) cells was used to determine which signaling pathways are involved in EGF-induced paxillin phosphorylation. Paxillin was phosphorylated on Tyr 31 and Tyr 118 after induction of migration by EGF in HCE cells. Specific inhibition of JNK activation by inhibitor SP600125 or overexpression of a dominant-negative JNK mutant not only blocked EGF-induced cell migration, but also eliminated tyrosine phosphorylation of paxillin on Tyr 31 and Tyr 118. HCE cells overexpressing paxillin-S178A mutant also exhibited lower mobility, and reduced phosphorylation of Tyr 31 and Tyr 118. However, paxillin-S178A-inhibited cell migration can be rescued by overexpression of paxillin-Y31E/Y118E mutant. Importantly, inhibition of JNK by SP600125 or overexpression of paxillin-S178A mutant prevented the association of FAK with paxillin. Taken together, these results suggest that phosphorylation of paxillin on Ser 178 by JNK is required for the association of paxillin with FAK, and subsequent tyrosine phosphorylation of paxillin.     

Multi-site phosphorylation of the protein tyrosine phosphatase, PTP1B: identification of cell cycle regulated and phorbol ester stimulated sites of phosphorylation.

The non-transmembrane protein tyrosine phosphatase, PTP1B, comprises 435 amino acids, of which the C-terminal 114 residues have been implicated in controlling both localization and function of this enzyme. Inspection of the sequence of the C-terminal segment reveals a number of potential sites of phosphorylation. We show that PTP1B is phosphorylated on seryl residues in vivo. Increased phosphorylation of PTP1B is seen to accompany the transition from G2 to M phase of the cell cycle. Two major tryptic phosphopeptides appear in two-dimensional maps of PTP1B from mitotic cells. One of these comigrates with the peptide generated following phosphorylation of PTP1B in vitro at Ser386 by the mitotic protein Ser/Thr kinase p34cdc2:cyclin B. The site of phosphorylation that is responsible for the pronounced retardation in the electrophoretic mobility of PTP1B from mitotic cells has been identified by site directed mutagenesis as Ser352. The identify of the kinase responsible for this modification is presently unknown. We also show that stimulation of HeLa cells with the phorbol ester TPA enhances phosphorylation of PTP1B. Two dimensional phosphopeptide mapping reveals that the bulk of the phosphate is in a single tryptic peptide. The site, identified as Ser378, is also the site of phosphorylation by protein kinase C (PKC) in vitro. Thus the TPA-stimulated phosphorylation of PTP1B in vivo appears to result directly from phosphorylation by PKC. The effect of phosphorylation on the activity of PTP1B has been examined in immunoprecipitates from TPA-treated and nocodazole-arrested cells. TPA treatment does not appear to affect activity directly, whereas the activity of PTP1B from nocodazole-arrested cells is only 70% of that from asynchronous populations.     

Ser/Thr/Tyr protein phosphorylation in bacteria - for long time neglected, now well established

The first clearly established example of Ser/Thr/Tyr phosphorylation of a bacterial protein was isocitrate dehydrogenase. In 1979, 25 years after the discovery of protein phosphorylation in eukaryotes, this enzyme was reported to become phosphorylated on a serine residue. In subsequent years, numerous other bacterial proteins phosphorylated on Ser, Thr or Tyr were discovered and the corresponding protein kinases and P-protein phosphatases were identified. These protein modifications regulate all kinds of physiological processes. Ser/Thr/Tyr phosphorylation in bacteria therefore seems to play a similar important role as in eukaryotes. Surprisingly, many bacterial protein kinases do not exhibit any similarity to eukaryotic protein kinases, but rather resemble nucleotide-binding proteins or kinases phosphorylating diverse low-molecular-weight substrates.     

Insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function.

We have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (Tyr1158, Tyr1162, and Tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. A single-Phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. The double-Phe replacements showed a 60-70% reduction, and substitution of all 3 tyrosine residues with Phe or Ser reduced insulin-stimulated tyrosine autophosphorylation by greater than 80%. Phosphopeptide mapping each mutant revealed that all remaining tyrosine autophosphorylation sites were phosphorylated normally following insulin stimulation, and no new sites appeared. The single-Phe mutants showed insulin-stimulated kinase activity toward a synthetic peptide substrate of 50-75% when compared with wild-type receptor kinase activity. Insulin-stimulated kinase activity was further reduced in the double-Phe mutants and barely detectable in the triple-Phe mutants. In contrast to the wild-type receptor, all of the mutant receptor kinases showed a significant reduction in activation following in vitro insulin-stimulated autophosphorylation. When studied in intact Chinese hamster ovary cells, insulin-stimulated receptor autophosphorylation and tyrosine phosphorylation of the cellular substrate pp185 in the single-Phe and double-Phe mutants was progressively lower with increased tyrosine replacement and did not exceed the basal levels in the triple-Phe mutants. However, all the mutant receptors, including the triple-Phe mutant, retained the ability to undergo insulin-stimulated Ser and Thr phosphorylation. Thus, full activation of the insulin receptor tyrosine kinase is dependent on insulin-stimulated Tris phosphorylation of the kinase domain, and the level of autophosphorylation in the kinase domain provides a mechanism for modulating insulin receptor kinase activity following insulin stimulation. By contrast, insulin stimulation of receptor phosphorylation on Ser and Thr residues by cellular serine/threonine kinases can occur despite markedly reduced tyrosine autophosphorylation.     

Involvement of the membrane distal catalytic domain in pervanadate-induced tyrosine phosphorylation of receptor protein-tyrosine phosphatase alpha

Receptor protein-tyrosine phosphatase alpha, RPTPalpha, is a typical transmembrane protein-tyrosine phosphatase (PTP) with two cytoplasmic catalytic domains. RPTPalpha became strongly phosphorylated on tyrosine upon treatment of cells with the PTP inhibitor pervanadate. Surprisingly, mutation of the catalytic site Cys in the membrane distal PTP domain (D2), but not of the membrane proximal PTP domain (D1) that harbors the majority of the PTP activity, almost completely abolished pervanadate-induced tyrosine phosphorylation. Pervanadate-induced RPTPalpha tyrosine phosphorylation was not restricted to Tyr789, a known phosphorylation site. Cotransfection of wild-type RPTPalpha did not potentiate tyrosine phosphorylation of inactive RPTPalpha-C433SC723S, suggesting that RPTPalpha-mediated activation of kinase(s) does not underlie the observed effects. Mapping experiments indicated that pervanadate-induced tyrosine phosphorylation sites localized predominantly, but not exclusively, to the C-terminus. Our results demonstrate that RPTPalpha-D2 played a role in pervanadate-induced tyrosine phosphorylation of RPTPalpha, which may suggest that RPTPalpha-D2 is involved in protein-protein interactions.     

Involvement of WalK (VicK) phosphatase activity in setting WalR (VicR) response regulator phosphorylation level and limiting cross‐talk in Streptococcus pneumoniae D39 cells

WalRK (YycFG) two‐component systems (TCSs) of low‐GC Gram‐positive bacteria play critical roles in regulating peptidogylcan hydrolase genes involved in cell division and wall stress responses. The WalRK (VicRK) TCSs of Streptococcus pneumoniae (pneumococcus) and other Streptococcus species show numerous differences with those of other low‐GC species. Notably, the pneumococcal WalK sensor kinase is not essential for normal growth in culture, unlike its homologues in Bacillus and Staphylococcus species. The WalK sensor kinase possesses histidine autokinase activity and mediates dephosphorylation of phosphorylated WalR～P response regulator. To understand the contributions of these two WalK activities to pneumococcal growth, we constructed and characterized a set of walK kinase and phosphatase mutants in biochemical reactions and in cells. We identified an amino acid substitution in WalK that significantly reduces phosphatase activity, but not other activities. Comparisons were made between WalRK regulon expression levels and WalR～P amounts in cells determined by Phos‐tag SDS‐PAGE. Reduction of WalK phosphatase activity resulted in nearly 90% phosphorylation to WalR～P, consistent with the conclusion that WalK phosphatase is strongly active in exponentially growing cells. WalK phosphatase activity was also shown to depend on the WalK PAS domain and to limit cross‐talk and the recovery of WalR～P from walK⁺ cells.     

Agents of change – concepts in Mycobacterium tuberculosis Ser/Thr/Tyr phosphosignalling

The flow of information from the outside to the inside of bacterial cells is largely directed by protein kinases. In addition to histidine/aspartate phosphorelays of two‐component response regulators, recent work in Mycobacterium tuberculosis (Mtb) reinforces the idea that phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) is central to bacterial physiology and pathogenesis, and that the corresponding phosphosystems are highly similar to those in eukaryotes. In this way, eukaryotes are a useful guide to understanding Ser/Thr/Tyr phosphorylation (O‐phosphorylation) in prokaryotes such as Mtb. However, as novel functions and components of bacterial O‐phosphorylation are identified, distinct differences between pro‐ and eukaryotic phosphosignalling systems become apparent. The emerging picture of O‐phosphorylation in Mtb is complicated, goes beyond the eukaryotic paradigms, and shows the limitations of viewing bacterial phosphosignalling within the confines of the ‘eukaryotic‐like’ model. Here, we summarize recent findings about Ser/Thr and the recently discovered Tyr phosphorylation pathways in Mtb, highlight the similarities and differences between eukaryotic and prokaryotic O‐phosphorylation, and pose additional questions about signalling components, pathway organization, and ultimately, the cellular roles of O‐phosphorylation in Mtb physiology and pathogenesis.     

Helicobacter pylori CagA containing ITAM-like sequences localized to lipid rafts negatively regulates VacA-induced signaling in vivo

Background. Helicobacter pylori CagA is injected into the host cell and tyrosine‐phosphorylated. We examined tyrosine‐phosphorylation sites of CagA, as well as the function of CagA proteins in vivo and in vitro. Methods. After proteolytic digestion of CagA with lysyl endopeptidase, CagA tyrosine‐phosphorylation sites were determined using quadropolar time‐of‐flight (Q‐TOF) mass spectrometry analysis. Specific anti‐pY CagA polyclonal and anti‐CagA monoclonal antibodies were used to examine gastric mucosal biopsy specimens from H. pylori infected patients. Results. Mass spectrometry identified five crucial tyrosine‐phosphorylation sites of CagA at Tyr893, Tyr912, Tyr965, Tyr999, and Tyr1033 within the five repeated EPIYA sequences of H. pylori (NCTC11637)‐infected AGS cells. CagA protein also had an immuno‐receptor tyrosine‐based activation motif (ITAM)‐like amino acid sequences in the 3′ region of the cagA, E PIY ATI x₂₇EIY ATI , which closely resembled the ITAM. CagA proteins: (i) were localized to the 1% TritonX‐100 resistant membrane fraction (lipid rafts); (ii) formed a cluster of phosphorylated CagA protein complexes; (iii) associated with tyrosine‐phosphorylated GIT1/Cat1 (G protein‐coupled receptor kinase‐interactor 1/Cool‐associated tyrosine‐phosphorylated 1), substrate molecules of receptor type protein‐tyrosine phosphatase (RPTPζ/β), which is the receptor of VacA; and (iv) were involved in a delay and negative regulation of VacA‐induced signal. Furthermore, immunohistochemical staining of gastric mucosal biopsy specimens provided strong evidence that tyrosine‐phosphorylated CagA is found together with CagA at the luminal surface of gastric foveola in vivo. Conclusion. These findings suggest an important role for CagA containing ITAM‐like sequences in the pathogenesis of H. pylori‐related disease.     

Phosphoproteome analysis of the pathogenic bacterium Helicobacter pylori reveals over-representation of tyrosine phosphorylation and multiply phosphorylated proteins

Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues is a major regulatory post-translational modification in the bacteria. To reveal the phosphorylation state in the Gram-negative pathogenic bacterium Helicobacter pylori, we carried out a global and site-specific phosphoproteomic analysis based on TiO(2) -phosphopeptide enrichment and high-accuracy LC-MS/MS determination. Eighty-two phosphopeptides from 67 proteins were identified with 126 phosphorylation sites, among which 79 class I sites were determined to have a distribution of 42.8:38.7:18.5% for the Ser/Thr/Tyr phosphorylation, respectively. The H. pylori phosphoproteome is characterized by comparably big size, high ratio of Tyr phosphorylation, high abundance of multiple phosphorylation sites in individual phosphopeptides and over-representation of membrane proteins. An interaction network covering 28 phosphoproteins was constructed with a total of 163 proteins centering on the major H. pylori virulence factor VacA, indicating that protein phosphorylation in H. pylori may be delicately controlled to regulate many aspects of the metabolic pathways and bacterial virulence.     

Regulation of Dictyostelium protein-tyrosine phosphatase-3 (PTP3) through osmotic shock and stress stimulation and identification of pp130 as a PTP3 substrate

Osmotic shock and growth-medium stimulation of Dictyostelium cells results in rapid cell rounding, a reduction in cell volume, and a rearrangement of the cytoskeleton that leads to resistance to osmotic shock. Osmotic shock induces the activation of guanylyl cyclase, a rise in cGMP mediating the phosphorylation of myosin II, and the tyrosine phosphorylation of actin and the approximately 130-kDa protein (p130). We present data suggesting that signaling pathways leading to these different responses are, at least in part, independent. We show that a variety of stresses induce the Ser/Thr phosphorylation of the protein-tyrosine phosphatase-3 (PTP3). This modification does not alter PTP3 catalytic activity but correlates with its translocation from the cytosol to subcellular structures that co-localize to endosomal vesicles. This translocation is independent of PTP3 activity. Mutation of the catalytically essential Cys to a Ser results in inactive PTP3 that forms a stable complex with tyrosine-phosphorylated p130 (pp130) in vivo and in vitro, suggesting that PTP3 has a substrate specificity for pp130. The data suggest that stresses activate several interacting signaling pathways controlled by Ser/Thr and Tyr phosphorylation, which, along with the activation of guanylyl cyclase, mediate the ability of this organism to respond to adverse changes in the external environment.